Convergent evolution of SARS-CoV-2 XBB lineages on receptor-binding domain 455–456 synergistically enhances antibody evasion and ACE2 binding

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) XBB lineages have achieved dominance worldwide and keep on evolving. Convergent evolution of XBB lineages on the receptor-binding domain (RBD) L455F and F456L is observed, resulting in variants with substantial growth advantages, such as EG.5, FL.1.5.1, XBB.1.5.70, and HK.3. Here, we show that neutralizing antibody (NAb) evasion drives the convergent evolution of F456L, while the epistatic shift caused by F456L enables the subsequent convergence of L455F through ACE2 binding enhancement and further immune evasion. L455F and F456L evade RBD-targeting Class 1 public NAbs, reducing the neutralization efficacy of XBB breakthrough infection (BTI) and reinfection convalescent plasma. Importantly, L455F single substitution significantly dampens receptor binding; however, the combination of L455F and F456L forms an adjacent residue flipping, which leads to enhanced NAbs resistance and ACE2 binding affinity. The perturbed receptor-binding mode leads to the exceptional ACE2 binding and NAb evasion, as revealed by structural analyses. Our results indicate the evolution flexibility contributed by epistasis cannot be underestimated, and the evolution potential of SARS-CoV-2 RBD remains high.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been continuously circulating and evolving worldwide [1][2][3][4].Since late 2022, XBB* variants, especially XBB.1.5 and other XBB derivatives with a proline on residue 486 (486P) on the receptor-binding domain (RBD) of the virus Spike glycoprotein (S) started to dominate, which demonstrated enhanced binding to human ACE2 while maintaining extremely strong capability of evading humoral immunity [5][6][7][8].These immune-evasive lineages are still continuously accumulating more S mutations, such as R403K, V445S, L455F, F456L and K478R, that may lead to further shift in antigenicity and escape from neutralizing antibodies elicited by repeated vaccination and infection [9,10].Some immune escape mutations, represented by F456L, even convergently appeared recently in multiple independent XBB derivative strains, such as EG.5, XBB.1.5.10,FE.1 and FD.1.1,indicating strong selection pressure due to herd immunity (Fig 1A) [11,12].By October 2023, over 70% of newly uploaded SARS-CoV-2 sequences carry F456L mutation.Furthermore, multiple independent XBB lineages with both F456L and L455F are growing rapidly in different countries, such as XBB.1.5.70/GK.* in Brazil, the United States, and Canada, and HK.3 (EG.5.1.1.3)in China (Fig 1B and 1C) [12][13][14].However, the proportion of lineages with L455F mutation but without F456L is extremely low, exhibiting no growth advantage (Fig 1C).Interestingly, L455 and F456 are two adjacent residues on the receptorbinding motif (RBM) of SARS-CoV-2 RBD, and the variant is just the "flipping" of the two residues, L455-F456 to F455-L456, also known as the "FLip" mutant (Fig 1B).These two sites are also located on a critical epitope that targeted by the public IGHV3-53/3-66 Class 1 NAbs [15][16][17].Mutations on these sites are likely to escape this type of NAbs that are abundant in vaccinated and convalescent individuals, leading to substantial reduction of protection efficiency [18,19].It is crucial to investigate the impacts on immune evasion and infection efficiency, especially for recent convalescents who recovered from XBB breakthrough infections, and the underlying mechanism of such synergistic effects that enables the unexpected advantage of L455F mutation on the basis of XBB*+F456L lineages, to explain the exceptional growth advantage of such lineages.

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Convergent evolution of XBB RBD 455-456 synergistically enhances antibody evasion and ACE2 binding

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Convergent evolution of XBB RBD 455-456 synergistically enhances antibody evasion and ACE2 binding antibody evasion.Therefore, L455F further enhances the NAb evasion of XBB.1.5+F456Lvariants, which may contribute to the growth advantage of the "FLip" mutants.These findings indicate that L455F and F456L complement each other to achieve maximum capability of escaping NAbs, despite their both linear and spatial adjacency on the RBD.

L455F, F456L, and their combination specifically escape the majority of Class 1 NAbs
As residues 455 and 456 on RBD are mainly recognized by Class 1 antibodies, which is also referred to as "Group A1/A2" in our previous study, we tested the pseudovirus-neutralizing activities of a panel of XBB.1.5-effectiveRBD-targeting monoclonal NAbs against these newlyemerged XBB subvariants, which were isolated in previous studies [9,18] S4B).We also evaluated the activity of neutralizing mAbs reported as drug candidates, including three that had been approved for emergency use.Two of three tested Class 1 NAbs, BD57-0129 and Omi-42, were partially evaded by L455F and L455F+F456L, while BD56-1854 remains potent [26].The activity of Class 3 antibody S309 (Sotrovimab) is not substantially affected, remaining weak neutralization [27].As expected, SD1-targeting antibody S3H3, and our previously reported Class 1/4 (Group F3) RBD-targeting therapeutic neutralizing antibody SA55 remain potent against all tested XBB subvariants, given that the mutated residues in the variants are not directly recognized by SA55 and S3H3 (Fig 3D) [28,29].

Epistatic interactions of L455F and F456L on receptor binding affinity
The above results demonstrate that both the L455F and F456L mutations can confer significant resistance to neutralization by convalescent plasma, primarily mediated by escaping Class 1 NAbs.However, it remains unclear why, during natural evolution, various lineages consistently evolve F456L independently, followed by subsequent occurrence of the L455F mutation, while cases where L455F is acquired first are rarely observed.Due to the fact that residues 455-456 are also located on the RBM of RBD, we hypothesize that the effects of the L455F and F456L combination originate from their impact on the affinity to the cell surface receptor for virus entry, human ACE2 (hACE2).Previous DMS data have indicated that individual substitutions, L455F or F456L, both lead to a substantial decrease in hACE2 affinity within the BA.2 background [30].However, as the combination of these two mutations essentially results in an adjacent residue flipping, there might be a local compensatory effect on hACE2 binding.The significant antigenic shift from BA.2 to XBB.1.5 may also alter the impacts of the two mutations on receptor binding.To validate the hypothesis, we constructed recombinant RBD subunits of XBB.1.5,XBB.1.5+L455F,XBB.1.5+F456L,and XBB.1.5+L455F+F456L("FLip"), and determined their binding affinities to hACE2 by surface plasmon resonance (SPR) assays.The dissociation equilibrium constants (K D ) demonstrate 6.4 nM, 25 nM, 11 nM, and 2.0 nM for the four mutants, respectively (Fig 4A).Consistent with DMS results, L455F significantly dampens hACE2-binding affinity of XBB.1.5RBD, and F456L also slightly weakens the

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Convergent evolution of XBB RBD 455-456 synergistically enhances antibody evasion and ACE2 binding binding to hACE2.Surprisingly, although neither of the two mutations increases hACE2 affinity alone, their combination XBB.1.5+L455F+F456Lexhibits significantly higher affinity than XBB.1.5.As for the kinetics, L455F or F456L alone does not largely affect the association kinetic constant (k a ), but L455F greatly fastens the dissociation (k d ) (S5 Fig) .In contrast, "FLip" not only rescues the accelerated dissociation, but also facilitates association, synergistically improving the receptor binding at both thermodynamic and kinetic levels (Figs 4B and S5).

Structural basis of the enhanced ACE2-binding affinity and antibody evasion in "FLip" variants
To elucidate the disparities in binding affinity between XBB.1.5,XBB.1.5+F456L,and XBB.1.5+L455F+F456L ("FLip") spike proteins with ACE2, we determined the structures of these three spike proteins individually in complex with hACE2 to interrogate the conformational change on their RBD-ACE2 binding interface.As we expected, all three spike trimers mainly showed two 'RBD-open' and one 'RBD-closed' conformations with one or two ACE2 bound (Fig 5A).To further reveal the impacts of mutations of residues 455 and 456 on the interface, we determined the high-resolution structures of these three variants' RBD in complex with ACE2 at resolution of 3.3 Å, 3.0 Å, and 3.0 Å, respectively (Fig 5B ).With an unambiguous electron density observed, reliable analyses of the interaction interface can be performed (S6 Fig).
Compared to XBB.1.5,F456L mutation does not substantially affect the interactions on the RBD-ACE2 interface.Although the side chain size of leucine is smaller than phenylalanine, which should slightly weaken the hydrophobic packing, the packing among RBD-L455, RBD-F456, RBD-Y489, and ACE2-F28/D30/K31 is largely kept in L456 mutant, which is in line with the slightly reduced ACE2-binding affinity of F456L (Figs 5C and S7).In the case of "FLip" (L455F+F456L), the F455 manifests a distinct side-chain orientation and conformation compared to L455 (Fig 5D).This unique pattern confers more flexible space for Q493 on RBD and the H34 on ACE2, hence enabling insertion of H34 side chain between RBD Q493 and S494, which cannot be realized in XBB.1.5 or XBB.1.5+F456L,due to potential clash between L455 and Q493 in this conformation (Fig 5E).Consequently, two additional hydrogen bonds, H34-Q493 (3.57Å) and H34-S494 (2.57Å), are introduced, enhancing the affinity of "FLip" .Further analysis concludes that F456L is a prerequisite for the fitness of "FLip".Superimposition of F456 onto "FLip" RBD could form pronounced steric clashes between F455 and F456, thereby disturbing the binding mode of "FLip" RBD-ACE2 interface (Fig 5E).Above all, the flip of L455 and F456 lead to synergistic effect of residues around and reorganize the interface between "FLip" RBD and ACE2 so that enhanced binding affinity can be obtained.
In parallel, to investigate the mechanism of L455F/F456L capability of escaping Class 1 NAbs, we conducted an analysis of the impact of the "FLip" mutation on the alteration of the neutralizing activity of NAb Omi-42, which efficiently neutralizes XBB.1.5but not "FLip" variants [26,31,32].Published structural models of Omi-42 in complex of Beta Spike (PDB:7ZR7) revealed that residues 455 and 456 are critically recognized by its heavy chain (Fig 6A).The superimposition of three RBDs onto the structure of mAb Omi-42 in complex with Beta RBD reveals a slight reduction in the hydrophobic patch of XBB.1.5+F456L,while Y110 of Omi-42 CDR-H3 cannot be accessible to engage in hydrophobic interaction in "FLip" RBD, resulting in a weaker interaction, consistent with the previous conclusion (Fig 6B and 6D).Notably, different Class 1 NAbs could exhibit significantly distinct interaction patterns between RBD, especially L455/F456 region, since this region is generally targeted by the highly variable CDR-H3 loop of most Class 1 NAbs, especially the public IGHV3-53/3-66 antibodies [16,17,33].

Discussion
In this study, we evaluated the impacts of L455F and F456L, two frequently emerging adjacent mutations that convergently occur in multiple XBB sublineages, on antibody neutralization and receptor binding.We demonstrate that L455F further evades Class 1 NAbs on the XBB.1.5+F456L basis, while single F456L or L455F substitution dampens ACE2 binding.Surprisingly, their combination, which is exactly a "FLip", i.e. "Leu-Phe" to "Phe-Leu" flipping, between adjacent residues on the ACE2 binding interface, dramatically enhances affinity to ACE2.Together, these results explain the convergent evolution of XBB subvariants which evolve F456L and L455F in succession.Epistasis is a genetic phenomenon that the effect of one mutation is dependent on the presence of other mutations, resulting in non-additive impacts of mutations on specific functions [34,35].Epistatic effects on the fitness of several evolving epidemic viruses, such as influenza, have been described by experiments, including DMS [36,37].The epistatic interaction between two early SARS-CoV-2 RBD mutations on RBD, Q498R and N501Y has also been reported in a previous study [38].Based on the ancestral SARS-CoV-2 RBD, Q498R alone slightly reduced ACE2 binding affinity, while a strong enhancement in affinity is observed for Q498R based on N501Y (RBD of Alpha VOC).The epistatic shift described here is even more striking.L455F caused strong affinity reduction on XBB.1.5,but significantly improved the hACE2-binding affinity of XBB.1.5+F456L.Both L455 and F456 are on the core of the RBD-hACE2 interface, forming a compact conformation with D30 and K31 on ACE2 [39].Our structural analyses reveal that "FLip" mediates substantial conformation change on the RBD-hACE2 binding interface, which involves the remodeling of not only 455/ 456, but also Q493 on RBD and H34 on hACE2.In prior Omicron lineages, such as BA.1 and BA.2, the substitution of glutamine (Q) with arginine (R) at residue 493 on RBD significantly diminished ACE2 affinity [9,40].A recent DMS study demonstrated that F456L is much more deleterious to ACE2 binding on R493 backbone (BA.1/BA.2) than Q493 backbone (XBB.1.5)[41].Our structural analyses show that in the Q493 background (XBB.1.5),F456L provides a space for insertion of the side chain of K31 into the RBD, consequently introducing a hydrogen bond between K31 of ACE2 and backbone of S490 of RBD to compensate for the dampened hydrophobic packing, which cannot be achieved in the background of R493 (BA.2) due to the steric clash between ACE2-K31 and RBD-R493, making the co-occurrence of F456L and R493 deleterious (S7B Fig) .It would be intriguing to investigate whether the "FLip" further accentuates the role of Q493, potentially resulting in further epistatic effects.
ACE2 mimicry has been considered to be a useful strategy for identifying and designing broad-spectrum SARS-CoV-2-neutralizing binders and antibodies [42,43].Class 1 NAbs, such as Omi-42, usually interact with RBD L455/F456 with heavy chain CDR3 in a distinct way compared to ACE2, despite the partially overlapped footprints on RBD (Fig 6A and 6B) [26].Similarly, most XBB.1.5-effectiveClass 1 NAbs failed to mimic the binding mode of ACE2 and escaped by the XBB.1.5+L455F+F456L"FLip" mutants.The emergence of L455F+F456L indicates that such receptor mimicry could be extremely difficult.To achieve the goal that mutants escaping the neutralizer also lose affinity to the receptor, the binder should not only target the same residues as those targeted by the receptor, but also mimic the binding mode of the receptor.To further investigate and explain such phenomenon, detailed analyses of the impacts these two mutations on other RBD background, particular for the variants with distinct antigenicity compared to XBB subvariants, such as BA.2.86, should be carefully evaluated in the future.
Overall, our work rationalized the emergence and circulation of XBB sublineages with F456L followed by L455F mutation, and highlighted the enhanced receptor-binding affinity and neutralizing antibody escape, which may lead to higher transmissibility and risk of breakthrough infection and reinfection.Considering the continuously increasing proportion of these variants, the efficacy of developing NAb drugs and vaccines against them should be carefully evaluated.Epistasis could substantially extend the possibility of accumulating mutations for SARS-CoV-2 RBD, leading to novel mutants with extremely high capability of escaping NAbs without great compromise on infectivity.Indeed, some additional immune-evasive mutations, including A475V, is convergently emerging on the basis of "FLip" strains.The evolutionary potential of SARS-CoV-2 RBD is still high, and should not be underestimated.

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Convergent evolution of XBB RBD 455-456 synergistically enhances antibody evasion and ACE2 binding Blood samples from convalescent patients who had recovered from SARS-CoV-2 Omicron BTI or reinfection were obtained under the study protocols approved by Beijing Ditan Hospital, Capital Medical University (Ethics committee archiving No. LL-2021-024-02) and the Tianjin Municipal Health Commission, and the Ethics Committee of Tianjin First Central Hospital (Ethics committee archiving No. 2022N045KY) (S1 Table ).Written informed consent, for the collection of information, storage and use of blood samples for research and data publication, was obtained from each participant.

Patient recruitment and plasma isolation
The infections of patients in the BA.5 or BF.7 BTI cohort were confirmed between July and October 2022, during the "zero COVID" period in China.These infections were confirmed by PCR, and the viral strains of the majority of them were determined sequencing.Other samples which were not sequenced also showed strong epidemiological correlations with the sequenced samples.
Whole blood samples were diluted 1:1 with PBS+2% FBS and then subjected to Ficoll (Cytiva, 17-1440-03) gradient centrifugation.After centrifugation, plasma was collected from the upper layer.Plasma samples were aliquoted and stored at −20 ˚C or less and were heatinactivated before experiments.
Huh-7 cell line (Japanese Collection of Research Bioresources [JCRB], 0403) was used in pseudovirus neutralization assays.Plasma samples or antibodies were serially diluted in culture media and mixed with pseudovirus, and incubated for 1 h in a 37˚C incubator with 5% CO 2 .Digested Huh-7 cells were seeded in the antibody-virus mixture.After one day of culture in the incubator, the supernatant was discarded.D-luciferin reagent (PerkinElmer, 6066769) was added into the plates and incubated in darkness for 2 min, and cell lysis was transferred to the

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Convergent evolution of XBB RBD 455-456 synergistically enhances antibody evasion and ACE2 binding detection plates.The luminescence value was detected with a microplate spectrophotometer (PerkinElmer, HH3400).IC50 was determined by a four-parameter logistic regression model.

Antibody expression and purification
Antibody heavy and light chain genes were synthesized by GenScript, separately inserted into vector plasmids (pCMV3-CH, pCMV3-CL or pCMV3-CK) by infusion (Vazyme), and cotransfected into Expi293F cells (Thermo Fisher) using polyethylenimine transfection.The transfected cells were cultured at 36.5˚C in 5% CO 2 and 175 rpm.for 6-10 days.The expression fluid was collected and centrifuged.After centrifugation, supernatants containing monoclonal antibodies were purified using Protein A magnetic beads (Genscript), and the purified samples were verified by SDS-PAGE.

Recombinant RBD expression and purification
DNA fragments that encode SARS-CoV-2 variant RBD (Spike 319-541) were codon-optimized for human cell expression and synthesized by Genscript.His-AVI tags were added at the end of the fragments.The fragments were then inserted into pCMV3 vector through infusion (Vazyme).The recombination products were transformed into E. coli DH5α competent cells (Tsingke).Colonies with the desired plasmids were confirmed by Sanger sequencing (Azenta) and cultured for plasmid extraction (CWBIO).293F cells were transfected with the constructed plasmids and cultured for 6 days.Products were purified using Ni-NTA columns (Changzhou Smart-lifesciences, SA005100) and the purified samples were verified by SDS-PAGE.

Surface plasmon resonance
SPR experiments were performed on the Biacore 8K (Cytiva).Human ACE2 with Fc tag was immobilized onto Protein A sensor chips (Cytiva).Purified SARS-CoV-2 variant RBDs were prepared in serial dilutions (6.25, 12.5, 25, 50, and 100nM) and injected over the sensor chips.The response units were recorded by Biacore 8K Evaluation Software 3.0 (Cytiva) at room temperature, and the raw data curves were fitted to a 1:1 binding model using Biacore 8K Evaluation Software 3.0 (Cytiva).

Protein expression and purification for Cryo-EM
and was mutated as previously described [45].To obtain the protein, the expression vector was transiently transfected into HEK293F cells grown in suspension at 37˚C in a rotating, humidified incubator supplied with 8% CO 2 and maintained at 130 rpm.After incubation for 72h, the supernatant was harvested, concentrated, and exchanged into the binding buffer by tangential flow filtration cassette.The S proteins were then separated by chromatography using resin attached with streptavidin and further purified by size exclusion chromatography using a Superose 6 10/300(GE Healthcare) in 20 mM Tris, 200mM NaCl, pH8.0.
The resolutions were evaluated on the basis of the gold-standard Fourier shell correction (threshold = 0.143) and evaluated by ResMap.All dataset processing workflows are shown in S6 Fig.

Structural model fitting and refinement
The atom models of the complex were first fitting the chain of the apo (PDB: 7XNQ) and Fab (heavy chain: 7E5Y, light chain: 7RU3) into the obtained cryo-EM density by Chimera.Then the structure was manually adjusted and corrected according to the protein sequences and density in Coot, real-space refinement was performed by Phenix.